Herpes viruses for immune modulation

ABSTRACT

An attenuated herpes virus which lacks a functional vhs gene or a functional equivalent thereof, but which has a functional UL43 gene or functional equivalent thereof, stimulates an immune response when dendritic cells are infected with the virus.

[0001] This is a continuation-in-part of application Ser. No.09/744,942, which is a U.S. national phase of PCT/GB99/02529, filed Aug.2, 1999, the entire contents of which is incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to attenuated herpes simplexviruses capable of efficiently infecting dendritic cells. It alsorelates to the use of such viruses in immunotherapy approaches to thetreatment of disease.

BACKGROUND TO THE INVENTION

[0003] Dendritic cells (DCs) are the most potent antigen presentingcells and are efficient at inducing responses even to antigens to whichthe immune system has become tolerant. Thus for tumour immunotherapy, inwhich an immune response is raised against a tumour, the use of DCs maybe ideal if they were made to present tumour specific antigens. DCsmight also be used to present antigens derived from infectious agents,such as bacteria, viruses or parasites, providing protective ortherapeutic vaccines for such diseases. However effective transfer ofantigens into DCs for any of these targets has proved the greatestproblem with this approach.

[0004] To provide a realistic chance of generating a therapeutic immuneresponse against a tumour antigen or other disease related antigen,several conditions have to be met. Firstly, it is necessary to identifymolecules whose expression is tumour or disease specific (or at leastselective), and which can therefore serve as the target for an immuneresponse. This task has proved very difficult for the majority of commontumours, but is solved in for example the case of cervical cancer by thepresence, in most cases, of the viral oncogenes E6 and E7, and for othertumours, good candidate antigens are beginning to be identified. Forexample the MUC-1 gene product is over expressed in a number of tumours,including 90% of ovarian cancers. Various other tumour associatedantigens have also been identified, any of which might be used in animmunotherapy treatment of cancer. Further tumor associated antigenswill no doubt continue to be discovered over time. Secondly, followingthe identification of the antigen/antigens, it is necessary to deliverthe antigens in an immunogenic form to the immune system. To generatethe cellular immune response critical for tumour rejection, this meansthe proteins must either be delivered inside the cytoplasm of a hostcell (a difficult task for high molecular weight protein antigens) orsynthesized by the host cells themselves after gene delivery or DNAimmunisation. Viral vectors which have been considered for this purposeinclude vaccinia, adenoviruses, or retroviruses.

[0005] The cell-type which is now widely recognised as providing theoptimal immune stimulus is the dendritic cell (DC; see for exampleGirolomoni and Ricciardi-Castagnoli, 1997). Indeed the DC appears to bethe only cell-type capable of stimulating a primary immune response invivo, and moreover has even been shown to be capable of breakingestablished tolerance in certain circumstances. A number of groups areexploring the use of DCs in autologous adoptive immunotherapy protocolsto stimulate immune responses against tumours in the hope that they mayshow a therapeutic effect. Such protocols involve culture and/orenrichment of DCs from peripheral blood, in vitro loading of DCs withantigen and reintroduction of the DCs to the patient or direct in vivoloading of DCs with antigen. However this approach has been hampered bythe absence of efficient means by which to load these cells withantigens. Recent work has however shown that presentation of antigens bypeptide pulsed DCs has produced anti-tumour responses in vivo (Celluzziet al., 1996; Zitvogel et al., 1996). As regard to viral vectors,retroviruses do not give high efficiency gene delivery to dendriticcells (Reeves et al., 1996; Aicher et al., 1997), and in our hands,unlike work reported by others (Arthur et al., 1997), adenoviruses onlygive low efficiency gene delivery.

[0006] We have previously tested and reported that herpes simplexviruses (HSV) can efficiently infect and deliver genes to dendriticcells (Coffin et al., 1998; WO 00/08191). HSV has a number of advantagesover other vector systems for this purpose, in that it can efficientlyinfect a wide variety of cell-types (including some very hard to infectwith other vector systems e.g. Dilloo et al., 1997; Coffin et al.,1998), is easy to manipulate, and can accept large DNA insertionsallowing the expression of multiple genes (reviewed by Coffin andLatchman 1996). Delivery of multiple antigens to dendritic cells ex vivofollowed by re-introduction into the body or direct administration ofantigens to dendritic cells in vivo may be particularly promisingapproaches to the treatment of some cancers and infectious diseases.

[0007] WO 00/08191 teaches that wild type herpes simplex viruses preventantigen processing occurring in infected dendritic cells and that herpesviruses that either lack both functional UL43 and vhs genes or containmutations that minimise immediate early gene expression are capable ofefficiently infecting dendritic cells without preventing antigenprocessing occurring in the infected cells.

SUMMARY OF THE INVENTION

[0008] We have now surprisingly found that disruption of the geneencoding the virion host shut-off protein (vhs) in HSV vectors enablesefficient dendritic cell activation to occur in HSV infected cells.Disruption of the UL43 gene is not also needed. It has previously beenshown that HSV infected dendritic cells usually do not become activatedeither by infection itself, or by other stimuli (Sallo et al 1999, Kruseet al 2000).

[0009] We have identified a previously unknown function of the vhsprotein in preventing dendritic cell activation. Dendritic cellactivation is defined as the up-regulation of certain cell surfacemarkers as compared to the non-activated state. These markers includeCD83 and CD86. Dendritic cell activation may be stimulated by treatmentwith lipopolysaccharide (LPS). LPS treatment of dendritic cells infectedwith HSV does not result in the up-regulation of CD83 or CD86. We haveshown that LPS treatment of dendritic cells infected with a mutant HSVin which vhs is inactivated but which have a functional UL43 geneup-regulates both CD83 and CD86. Up-regulation of CD83 and CD86 is notobserved following LPS treatment of dendritic cells infected withviruses comprising a functional vhs gene. Thus our results indicatethat, for transduced dendritic cells to maximally stimulate an immuneresponse following herpes virus infection, the gene encoding vhs shouldbe disrupted but the gene encoding UL43 need not be.

[0010] Our results also demonstrate a role for vhs in the pathogenesisof wild type herpes simplex viruses. HSV infects dendritic cells at ahigh efficiency and it would seem likely that the reason it has evolvedto do this as a part of its natural life-cycle is so that it canminimise a cell-mediated immune response which might otherwise prevent alatent HSV infection being efficiently established or result inclearance of the virus during repeated cycles of latency andreactivation. Dendritic cell activation is important in the stimulationof an effective cell-mediated immune response. Vhs is a virion proteinand so, whilst HSV genes are generally not expressed at high levels indendritic cells, the vhs protein would be delivered to the dendriticcell along with the incoming virus. Thus the novel function of vhs inpreventing activation of dendritic cells infected with HSV is likely tobe an important function of vhs in the HSV lifecycle following infectionof a human with HSV.

[0011] Accordingly, the present invention provides a method ofstimulating an immune response in a human or animal subject, whichmethods comprises administering to a subject in need thereof aneffective amount of an attenuated herpes virus which:

[0012] (i) lacks a functional vhs gene, or a functional equivalentthereof; and

[0013] (ii) comprises a functional UL43 gene, or functional equivalentthereof; such that dendritic cells are infected with said virus.

[0014] Preferably said virus is a human herpes simplex virus. Morepreferably, said virus is HSV1 or HSV2. The dendritic cells may beinfected in vitro or in vivo.

[0015] The virus may contain one or more additional mutation. Theadditional mutations preferably minimise the toxicity of the virus.Typically such mutations result in reduced or minimised immediate early(IE) gene expression. Prevention or reduction of IE gene expressionprevents or reduces virus replication. Such mutations include, forexample, inactivating mutations in the genes encoding ICP4, ICP27, ICP0and/or ICP22, preferably ICP27 and/or ICP4. An inactivating mutation inthe vmw65-encoding gene removing its transactivating function may alsobe included (e.g. vmw65 mutations as in Ace et al., 1989 or Smiley etal.1997). Preferably the additional mutations may also minimise theimmune response-inhibitory activity of the virus. Such mutations includeinactivation of the gene encoding ICP47.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the viral strains 1764/27-/4-,1764/27-/4-/pR20.5/vhs, 1764/27-/4-/pR20.5/vhs/HBS-Ag and wild type HSVstrain 17+.

[0017]FIG. 2 shows the results of FACS analysis to determine the levelsof cell-surface expression of CD40, CD80, CD83 and CD86 on un-stimulated(FIG. 2A) and LPS stimulated (FIG. 2B) mock infected cells and cellsinfected with 17⁺, 1764/27-/4- and 1764/27-/4-/pR20.5/vhs viral strains.

[0018]FIG. 3 shows the results of ELISA analysis to determine the effectof viral infection on IL-6 and TNFα secretion from uninfected dendriticcells and dendritic cells infected with 17⁺, 1764/27-/4- and1764/27-/4-/pR20.5/vhs viral strains.

[0019]FIG. 4 shows the proliferative responses of T-cells prepared fromhepatitis-B vaccinated and un-vaccinated individuals in response toHBS-Ag. Dendritic cells taken from each individual were eitheruntreated, mixed with recombinant HBS-Ag protein (HBSAg*), infected withthe control vector (1764/27-/4-/pR20.5/vhs HBSAg) or infected with thevector expressing HBS-Ag (1764/27-/4-/pR20.5/vhs/HBS-Ag) before mixingwith the T cells.

DETAILED DESCRIPTION OF THE INVENTION

[0020] A. Viruses

[0021] A virus of the invention is capable of infecting dendritic cellswithout preventing the infected dendritic cells from being activated.Preferably dendritic cells infected with a virus of the invention at amultiplicity of infection (MOI) of 1 can be activated by treatment withLPS or other activation stimuli.

[0022] A virus of the invention does not prevent the activation ofdendritic cells. To determine when a virus allows the activation ofdendritic cells to occur, dendritic cells are infected with the virus ata MOI≧1 and infected dendritic cells are treated with LPS. The levels ofcell surface markers such as CD83 and/or CD86 which are up-regulated onactivation of dendritic cells may be monitored to determine dendriticcell activation, for example by FACS analysis. The level of thesemarkers on the cell surface will be significantly higher in LPS treateddendritic cells than in cells which have not been treated with LPS ifthe virus with which the cells are infected allows dendritic cellactivation to occur. Some or all of these markers will also be higher ondendritic cells which have been infected with a virus that allowsdendritic cell activation to occur compared to uninfected dendriticcells. If a herpes simplex virus which does not contain an inactivatingmutation in vhs is used to infect dendritic cells, significantly lessup-regulation of these markers is observed.

[0023] To permit activation of infected dendritic cells to occur, avirus of the invention will lack a functional gene encoding vhs (in HSV)or homologues or functional equivalents thereof in other viral species.In addition, a virus of the invention will have a functional UL43 gene.Additional mutations may be made to reduce further theimmune-response-inhibitory effects of the virus for example by theinclusion of a mutation in the gene encoding ICP47.

[0024] An attenuated virus of the invention is preferably capable ofinfecting dendritic cells such that minimal toxicity results. Preferablycell survival post-infection will be at least 50% at one daypost-infection, more preferably at least 60, 70, 80 or 90% at one daypost-infection. To achieve reduced toxicity one or more mutations whichreduce viral replications may be included in a virus of the invention.For example, a mutation in the gene encoding VMW65 which mutationminimises the trans-activating activity of the protein and/or a mutationin one or more regulatory immediate early gene such as ICP4, ICP27, ICP0and ICP22 may be included. Thus a virus of the invention may typicallylack functional vhs, ICP27, ICP4 genes and comprise a VMW65 gene whichencodes a protein which does not exhibit transcriptional-activationactivity, or may typically lack functional vhs, ICP47 and ICP4 genes.

[0025] For direct use in vivo some degree of replication competence maytypically be beneficial in boosting the immune responses induced. Thusin these circumstances, a virus of the invention preferably lacks afunctional vhs gene and may also lack one or more functional genes whichare necessary for full pathogenicity of the virus but which are notnecessary for viral replication. Such genes include those encodingICP34.5, ICP6, thymidine kinase and glycoproteins such as gH.Preferably, however, the gene encoding thymidine kinase is functional asmutation of this gene would render the virus insensitive to anti-viralagents such as acyclovir.

[0026] Although the present invention has been exemplified using herpessimplex viruses, it will be understood that other viruses of theherpesviridae family may be modified to reduce the prevention ofdendritic cell activiation of infected dendritic cells. In particular,such viruses may include varicella zoster virus, pseudo-rabies virus orbovine herpes viruses.

[0027] When the virus of the invention is a herpes simplex virus, thevirus may be derived from, for example, HSV1 or HSV2 strains, orderivatives thereof, preferably HSV1. Derivatives include inter-typerecombinants containing DNA from HSV1 and HSV2 strains. Such inter-typerecombinants are described in the art, for example in Thompson et al(1988) and Meignier et al (1988). Derivatives preferably have at least70% sequence homology to either the HSV1 or HSV2 genomes, morepreferably at least 80%, even more preferably at least 90 or 95%,typically as measured by the methods described herein. More preferably,a derivative has at least 70% sequence identity to either the HSV1 orHSV2 genome, more preferably at least 80% identity, even more preferablyat least 90%, 95% or 98% identity.

[0028] A derivative may have the sequence of a HSV1 or HSV2 genomemodified by nucleotide substitutions, for example from 1, 2 or 3 to 10,25, 50 or 100 substitutions. The HSV1 or HSV2 genome may alternativelyor additionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends.

[0029] Derivatives which may be used to obtain the viruses of thepresent invention include strains that already have mutations in geneswhich it is desired to functionally inactivate in a virus of theinvention, for example vhs inactivated strains (as in Jones et al.1995),ICP47 inactivated strains (as in Goldsmith et al. 1998), strain d120which has a deletion in ICP4 (DeLuca et al., 1985), strain d27-1 (Riceand Knipe, 1990) which has a deletion in ICP27) or strain d92 which hasdeletions in both ICP27 and ICP4 (Samaniego et al., 1995). Use of thesestrains will reduce the number of steps required to produce the mutantHSV strains of the present invention.

[0030] The terminology used in describing the various HSV genes is asfound in Coffin and Latchman, 1996.

[0031] Where gene homologues of the HSV genes described above exist inother herpes virus species, then these homologues will be modified. By a“homologue” it is meant a gene which is functionally equivalent to a HSVgene a homologue typically exhibits sequence homology, either amino acidor nucleic acid sequence homology, to the corresponding HSV gene.Typically, a homologue of an HSV gene will be at least 15%, preferablyat least 20%, more preferably at least 30%, 40% or 50% identical at theamino acid level to the corresponding HSV gene.

[0032] The geen encoding vhs is the UL41 gene in HSV1 and HSV2. In HSV1strain 17+ (EMBL accession No. HELCG) the UL41 gene is from nucleotide91,170 to nucleotide 92,637. In HSV2 strain HG52 (EMBL accession No.z86099) the UL41 gene is from nucleotide 91,800 to nucleotide 93,275.

[0033] Methods of measuring nucleic acid and protein homology are wellknown in the art. For example the UWGCG Package provides the BESTFITprogram which can be used to calculate homology (for example used on itsdefault settings) (Devereux et al. (1984) Nucleic Acids Research 12,p387-395). The PILEUP and BLAST algorithms can be used to calculatehomology or line up sequences (typically on their default settings), forexample as described in Altschul (1993) J. Mol. Evol. 36:290-300;Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0034] Software for performing BLAST analyses is publicly availablethrough the National Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al., 1990). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

[0035] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a sequence is considered similar to another sequence if thesmallest sum probability in comparison of the first sequence to thesecond sequence is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0036] Homologues of HSV genes can be identified in a number of ways,for example by probing genomic or cDNA libraries made from other viruseswith probes comprising all or part of the HSV gene under conditions ofmedium to high stringency (for example 0.03M sodium chloride and 0.03Msodium citrate at from about 50° C. to about 60° C.). Alternatively,species homologues may also be obtained using degenerate PCR which willuse primers designed to target sequences within the variants andhomologues encoding conserved amino acid sequences. The primers willcontain one or more degenerate positions and will be used at stringencyconditions lower than those used for cloning sequences with singlesequence primers against known sequences (for example 0.03M sodiumchloride and 0.03M sodium citrate at about 40° C.).

[0037] A homologue in a herpes virus is a functional equivalent of anHSV protein if it shares one ore more functional characteristics withthe HSV protein. For example, a vhs protein plays a role in reducingprotein expression levels in an infected cell by reducing the stabilityof mRNA. Therefore, a functional equivalent of vhs protein preferablyplays a role in shutting down host-cell gene expression by reducing thestability of mRNA. More preferably, a functional equivalent of vhsprevents dendritic cell activation in response to stimuli which activateun-infected dendritic cells.

[0038] For reasons of safety, the viruses of the invention areattenuated, typically so that they are incapable of causing diseaseViral regions altered for the purposes of attenuation may be eithereliminated (completely or partly), or made non-functional, orsubstituted by other sequences, in particular by a heterologous genesequence. Attenuating mutations have been described for all viral groupsused as viral vectors. For example, HSV may be rendered avirulent bymutations in ICP34.5 and/or essential genes such as ICP4,ICP27 and/orthe vhs gene itself.

[0039] Particularly preferred attenuated viruses include viruses which,in addition to lacking a functional gene encoding vhs and optionallylacking a functional ICP47 gene, lack a functional ICP34.5 gene and afunctional ICP27 gene and optionally lacks a functional ICP4 gene and/ora VMW65 gene which encodes a protein which hastranscriptional-activation activity, and viruses which have a functionalICP27 gene but lack a functional ICP4 gene and a functional ICP34.5 geneand optionally lacks a VMW65 gene which encodes a protein which hastranscriptional-activation activity. Such viruses are described inWO98/04726 and WO99/60145, the disclosures of which are hereinincorporated by reference.

[0040] When a herpes simplex virus of the invention lacks a particularfunctional essential gene, for example a gene encoding ICP4 or ICP27,the virus is propagated using a cell line expressing that essentialgene. For example, when the virus lacks a functional ICP27 gene, thevirus may be propagated using V27 cells (Rice and Knipe, 1990), 2-2cells (Smith et al., 1992) or B130/2 cells (WO98/30707), preferablyB130/2 cells. When the virus lacks a functional ICP4 gene the virus maybe propagated using a cell line expressing ICP4, for example E5 cells(DeLuca et al., 1985). When the virus lacks a functional ICP4 gene and afunctional ICP27 gene the virus is propagated using a cell lineexpressing both ICP4 and ICP27 (such as E26 cells; Samaniego et al.,1995), and when the virus additionally lacks a functional vmw65 gene thevirus may be propagated using a cell line also containing a non-HSVhomologue of vmw65 (e.g. equine herpes virus gene 12 or BTIF from bovineherpes virus).

[0041] B. Methods of Mutation

[0042] The various viral genes referred to may be rendered functionallyinactive by several techniques well known in the art. For example, theymay be rendered functionally inactive by deletion(s), substitution(s) orinsertion(s), preferably by deletion. A deletion may remove portions ofa gene or the entire gene. For example, deletion of only one nucleotidemay be made, resulting in a frame shift. However, preferably largerdeletions are made, for example from 2, 3 or 5 to 10, 20, 30, 50, 100 or200 nucleotide substitutions. Preferably at least 25%, more preferablyat least 50% of the total coding and non-coding sequence (oralternatively, in absolute terms, at least 10 nucleotides, morepreferably at least 100 nucleotides, most preferably, at least 1000nucleotides) is deleted or substituted. It is particularly preferred toremove the entire gene and some of the flanking sequences. Insertedsequences may include the heterologous genes described below. Mutationsmay comprise both deletion(s) and insertion(s). For example, aninsertion may be made into the site of a deletion. Thus insertion of aheterologous gene into a viral gene may replace part or all of the viralgene. In particular, it is preferred to insert the heterologous geneinto vhs, ICP47, ICP27 or ICP4. In the case of the VMW65 gene, theentire gene is not deleted since it encodes an essential structuralprotein, but an inactivating mutation is typically made which abolishesthe ability of VMW65 to activate transcriptionally IE genes (e.g. as inAce et al., 1989 or Smiley et al., 1997).

[0043] Mutations may be made in the herpes viruses by homologousrecombination methods well known to those skilled in the art. Forexample, HSV genomic DNA is transfected together with a vector,preferably a plasmid vector, comprising the mutated sequence flanked byhomologous HSV sequences. The mutated sequence may comprise deletions,insertions or substitutions, all of which may be constructed by routinetechniques. Insertions may include selectable marker genes, for examplelacZ or GFP, for screening recombinant viruses by, for example,β-galactosidase activity or fluorescence.

[0044] C. Heterologous Genes and Promoters

[0045] The viruses of the invention may be modified to carry aheterologous gene/genes. The term “heterologous gene” encompasses anygene. Although a heterologous gene is typically a gene not present inthe genome of a herpes virus, a herpes gene may be used provided thatthe coding sequence is not operably linked to the viral controlsequences with which it is naturally associated. The heterologous genemay be any allelic variant of a wild-type gene, or it may be a mutantgene. The term “gene” is intended to cover nucleic acid sequences whichare capable of being at least transcribed to produce an RNA molecule,which RNA molecule is preferably capable of being translated to producea polypeptide or to down-regulate gene expression levels by ananti-sense effect A virus of the invention may optionally include someor all of 5′ and/or 3′ transcribed but untranslated flanking sequencesnaturally, or otherwise, associated with the translated coding sequenceof a heterologous gene. It may optionally further include the associatedtranscriptional control sequences normally associated with thetranscribed sequences, for example transcriptional stop signals,polyadenylation sites and downstream enhancer elements.

[0046] The heterologous gene/genes may be inserted into the viral genomeby homologous recombination of HSV strains with, for example, plasmidvectors carrying the heterologous gene/genes flanked by HSV sequences.The heterologous gene/genes may be introduced into a suitable plasmidvector comprising herpes viral sequences using cloning techniqueswell-known in the art. The heterologous gene/genes may be inserted intothe viral genome at any location provided that the virus can still bepropagated. It is preferred that the heterologous gene/genes is insertedinto a gene resulting in attenuation of the virus. Heterologous genesmay be inserted at multiple sites within the virus genome.

[0047] The transcribed sequence of the heterologous gene/genes ispreferably operably linked to a control sequence permitting expressionof the heterologous gene/genes in dendritic cells, preferably mammaliandendritic cells, more preferably human dendritic cells. The term“operably linked” refers to a juxtaposition wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequence.

[0048] The control sequence comprises a promoter allowing expression ofthe heterologous gene/genes and a signal for termination oftranscription. The promoter is selected from promoters which arefunctional in mammalian, preferably human dendritic cells. Thepromoter/promoters may be derived from promoter sequences of eukaryoticgenes. For example, promoters may be derived from the genome of a cellin which expression of the heterologous gene is to occur, preferably amammalian dendritic cell or more preferably a human dendritic cell. Withrespect to eukaryotic promoters, they may be promoters that function ina ubiquitous manner (such as promoters of β-actin, tubulin) or,alternatively, a dendritic cell-specific manner. Viral promoters mayalso be used, for example the Moloney murine leukaemia virus longterminal repeat (MMLV LTR) promoter or other retroviral promoters, thehuman or mouse cytomegalovirus (CMV) IE promoters.

[0049] Expression cassettes and other suitable constructs comprising theheterologous gene/genes and control sequences can be made using routinecloning techniques known to persons skilled in the art (see, forexample, Sambrook et al., 1989, Molecular Cloning—a laboratory manual;Cold Spring Harbor Press).

[0050] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancer sequences(including elements of the HSV LAT region). Chimeric promoters may alsobe used comprising sequence elements from two or more differentpromoters described above, for example an MMLV LTR/LAT fusion promoter(Lokensgard et al., 1994) or promoters comprising elements of the LATregion (WO98/30707).

[0051] Heterologous genes will typically encode polypeptides oftherapeutic use. For example, to promote an immune response specificallyagainst a particular tumour, it will be desirable to transfect dendriticcells with a virus of the invention directing expression of a tumourantigen/antigens. A tumour antigen may be specific to a tumour cell,i.e. present in tumour cells but not in non-tumour cells, or it may bepresent at higher levels in that tumour cell than in a non tumour cellof that type, for example due to up regulation of expression of theantigen. This will be useful in cancer therapy since an infecteddendritic cell of the invention can be used to stimulate the host immunesystem to react to the tumour-specific or tumour-prevalentantigen/antigens resulting in tumour reduction/regression. Inparticular, it is preferred that the tumour antigen/antigens isexpressed on the surface of the tumour cell, for example a cell surfacereceptor or cell adhesion protein. Examples of tumour antigens includethe MUC-1 gene product (Gendler et al., 1990) which is over expressed ina number of tumours including ovarian cancers, human papillomavirusproteins E6 and E7 which are associated with cervical cancer. MART-I,MAGE-I, gp100 and tyrosinase in melanoma, PSA in prostate cancer, CEA ina number of different types of tumour and Her2neu in various cancersincluding breast cancer.

[0052] Heterologous genes may also encode a polypeptide which is capableof modifying an immune response, for example cytokines (such as α-, β-or γ-interferon, interleukins including IL-1, IL-2, tumour necrosisfactor, or insulin-like growth factors I or II) or otherimmunomodulatory proteins including chemokines such as RANTES andco-stimulatory molecules such as CD80, CD86, CD40 and CD40 ligand.

[0053] The heterologous gene may also encode a polypeptide/polypeptidesof pathogenic origin so that, for example, a dendritic cell infectedwith a virus of the invention can be used to stimulate the host immunesystem to produce an immune response to a pathogen, either prior toinfection or after infection of the host by the pathogen. Viruses foruse in vaccines may typically comprise heterologous genes that encodeantigenic polypeptide(s). Preferably such polypeptides of pathogenicorigin are derived from pathogenic organisms, for example parasites,bacteria or viruses. Examples of such antigenic polypeptides includehepatitis C virus antigens, hepatitis B surface or core antigens,papillomavirus antigens, HIV antigens and malaria antigens. Virusescomprising heterologous genes from pathogenic organisms may be used foreither or both therapeutic and prophylactic treatment.

[0054] Therapeutic applications may well require the administration ofmultiple genes. The expression of multiple genes may be advantageous forthe treatment of a variety of conditions. Herpes viruses are uniquelyappropriate as they do not have the limited packaging capabilities ofother viral vector systems. Thus multiple heterologous genes can beaccommodated within its genome. For example, from 2 to 6 genes may beinserted into the genome.

[0055] There are, for example, at least two ways in which this could beachieved. For example, more than one heterologous gene and associatedcontrol sequences could be introduced into a particular HSV straineither at a single site or at multiple sites in the virus genome. Itwould also be possible to use pairs of promoters (the same or differentpromoters) facing in opposite orientations away from each other, thesepromoters each driving the expression of a heterologous gene (the sameor different heterologous gene) as described above.

[0056] D. Dendritic Cells

[0057] Dendritic cells can be isolated/prepared by a number of means,for example they can either be purified directly from peripheral blood,or generated from CD34+ precursor cells for example after mobilisationinto peripheral blood by treatment with G-CSF, or directly from bonemarrow. From peripheral blood adherent precursors can be treated with aGM-CSF/IL-4 mixture (Inaba et al., 1992), or from bone marrownon-adherent CD34+ cells can be treated with GM-CSF and TNF-α (Caux etal., 1992). DCs can be routinely prepared from the peripheral blood ofhuman volunteers, similarly to the method of Sallusto and Lanzavecchia,1994, using purified peripheral blood mononeucleocytes (PBMCs) andtreating 2 hour adherent cells with GM-CSF and IL-4. These are thendepleted of CD 19+ B cells and CD3+, CD2+ T cells using magnetic beads(see Coffin et al., 1998). Other methods may also be used for thepreparation of dendritic cells.

[0058] E. Therapeutic Uses

[0059] Viruses of the invention, and dendritic cells infected withviruses of the invention may be used in methods of therapy. Inparticular, viruses of the invention, and dendritic cells infected withviruses of the invention, which express tumour antigens may be used inmethods of treating cancer. Specifically, the, viruses of the invention,and dendritic cells infected with viruses of the invention may be usedto inhibit the growth of various tumours in mammals, including humans,such as, for instance, ovarian, cervical and endometrial tumours andcarcinomas, for example mammary carcinoma, lung carcinoma, bladdercarcinoma and colon carcinoma. Other neoplasms whose growth may beinhibited include sarcomas, for example soft tissue and bone sarcomas,and hematological malignancies such as leukemias. Particular examples ofcancers which may be treated using viruses of the invention and/ordendritic cells infected with viruses of the invention which expresstumour antigens include melanomas, leukemias, cervical cancers andovarian cancers. A virus for use in treating cancer typically comprisesa heterologous gene encoding a tumour antigen. Administration of such avirus, or dendritic cells infected with such a virus, will typicallyresult in the generation of an immune response to the tumour antigen.

[0060] Viruses of the invention, and dendritic cells infected withviruses of the invention, may be used in methods of treating orpreventing pathogenic infections, for example parasitic, bacterial orviral infections. A virus for use in treating a pathogenic infectiontypically comprises a heterologous gene encoding an antigen from thepathogenic organism. Administration of such a virus, or dendritic cellsinfected with such a virus, will typically result in the generation ofan immune response to antigen from the pathogenic organism. Such viralinfections include herpes virus infections. Thus, a virus of theinvention may be used to induce immune responses to the virus itself,for example in the treatment or vaccination of HSV1 or HSV2 infection.Where a virus is intended for use in the treatment of HSV1 or HSV2, thevirus may optionally contain a heterologous gene, which heterologousgene encodes an HSV antigen (which is not under the control of itsnatural promoter) or an immunomodulatory molecule. The viruses/dendriticcells may be administered prior to infection to stimulate a protectiveimmune response in the host, or after infection to stimulate the hostimmune system to combat the infection.

[0061] F. Administration

[0062] The herpes viruses of the present invention may thus be used todeliver therapeutic genes to a human or animal in need of treatment.Delivery of therapeutic genes using the herpes viruses of the inventionmay be used to treat for example, malignancies and/or pathogenicinfections.

[0063] The viruses of the invention may be used in a patient, preferablya human patient, in need of treatment. A patient in need of treatment isan individual suffering from cancer, or a patient with a pathogenicinfection. The aim of therapeutic treatment is to improve the conditionof a patient. Typically therapeutic treatment using a virus of theinvention allieviates the symptoms of the cancer. A method of treatmentof cancer according to the invention comprises administering atherapeutically effective amount of a virus having a functional UL43gene and lacking a functional vhs gene to a patient suffering fromcancer such that the virus is present in dendritic cells in the patient.Administration of virus of the invention to an individual suffering froma tumour will typically kill the cells of the tumour thus decreasing thesize of the tumour and/or preventing spread of malignant cells from thetumour.

[0064] Typically therapeutic treatment of a pathogenic infection using avirus of the invention alleviates the symptoms of the infection andpreferably kills the pathogenic organism. A method of treatment of apathogenic infection according to the invention comprises administeringa therapeutically effective amount of a virus lacking a functional vhsgene to a patient with a pathogenic infection. Preferably the virusenters dendritic cells in the patient or dendritic cells which have beeninfected with the virus ex vivo are administered to the patient.Prophylactic treatment using a virus of the invention typically leads tothe production of antibodies against a tumour antigen or against anantigen from a pathogenic organism in a patient at risk of cancer or apathological infection. Typically a patient at risk of cancer may begenetically disposed thereto or may have been exposed to or be at riskof exposure to a carcinogen. Typically a patient at risk of a pathogenicinfection may be likely to be exposed to a pathogenic organism.

[0065] One method for carrying out therapy involves inserting thetherapeutic gene/genes into the genome of the herpes virus of theinvention, as described above, and then combining the resultantrecombinant virus with a pharmaceutically acceptable carrier or diluentto produce a pharmaceutical composition. Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. The composition may be formulated for parenteral, intramuscular,intravenous, intraperitoneal, subcutaneous or transdermaladministration. Subcutaneous or intraperitoneal administration ispreferred. Trans- or intradermal administration may be particularlypreferred.

[0066] Infection of dendritic cells with the virus of the invention maybe carried out in vivo by administration of a composition comprising thevirus to a patient. The pharmaceutical composition is administered insuch a way that the virus containing the therapeutic gene/genes, caninfect dendritic cells. The amount of virus administered is in the rangeof from 10⁴ to 10¹⁰ pfu, preferably from 10⁵ to 10⁸ or from 10⁵ to 10 ⁹pfu, more preferably about 10⁶ to 10⁸ pfu. When injected intra-dermallyor trans-dermally administered, for example using a needle-free device,typically from 10 μl to 1 ml, preferably from 100 μl to 1 ml of virus ina pharmaceutically acceptable suitable carrier or diluent or in aparticulate composition is administered.

[0067] Another method involves isolating/preparing dendritic cells fromperipheral blood or bone marrow and infecting the cells with the virusof the invention in vitro. Transduced dendritic cells are then typicallyadministered to the patient by intramuscular, intraperitoneal,subcutaneous or intravenous injection, or by direct injection into thelymph nodes of the patient, preferably by subcutaneous, intraperitonealor direct injection into the lymph nodes. Typically from 10⁵ to 10⁹transduced dendritic cells, preferably from 10⁶ to 10⁸ cells, morepreferably about 10⁷ cells are administered to the patient.

[0068] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage for anyparticular patient. The dosage may be determined according to variousparameters, especially according to, for example, the age, weight andcondition of the patient.

[0069] The following Examples illustrate the invention.

EXAMPLES

[0070] Materials and Methods

[0071] Construction and Growth of Viral Strains

[0072] All virus strains are derived from HSV1 strain 17+, thenucleotide sequence of which is deposited in GenBank (Accession No.HE1CG). Viral strains were produced and propagated using BHK C-21 cells(ECACC No. 8501143) or BHK cells stably transfected with the genesencoding HSV1 ICP27, ICP4 and equine herpes virus gene 12 (Thomas et al.1999).

[0073] For viruses with mutations in VMW65, 3 mMhexamethylene-bisacetamide (HMBA) was included in the media used forvirus growth (McFarlane et al., 1992). The following viral strains wereused.

[0074] (i) 17+(Wild Type HSV1)

[0075] (ii) 17+/pR20.5/UL43

[0076] A cassette from plasmid pR20.5 (Thomas et al. 1999b) consistingof an RSV/lacZ/pA sequence and a CMV/GFP/pA sequence in oppositeback-to-back orientations and separated by an HSV LAT region sequence(nts 118,866-120,219) was inserted into the UL43 locus by homologousrecombination with purified genomic HSV1 strain 17+ DNA by standardmethods. The pR20.5 cassette was first inserted into a plasmidcontaining UL43 flanking regions (Coffin et al, 1996) at the unique NsiIsite, giving plasmid pR20.5/43. The 20.5 cassette can be excised fromits pGEM5 (Promega) plasmid backbone-with SrfI as an oligonucleotideencoding SrfI was inserted on either side of the cassette. The RSVpromoter was excised from pRc/RSV (Invitrogen), lacZ/pA from pCH110(Pharmacia), CMV/pA from pcDNA3 (Invitrogen) and GFP from pEGFP-N1(Clontech) for the construction of plasmid pR20.5.

[0077] (iii) 1764/27-/4-

[0078] Virus strain 1764/27-/4- was constructed by recombination ofvirus strain 1764/27-/4-/pR20.5 DNA with empty ICP4 flanking regions andthe selection of virus plaques which do not express GFP or lacZ. Virusstrain 1764/27-/4-/pR20.5 is described in Thomas et al. 1999b andcontains the pR20.5 cassette inserted into the ICP4 geneso as to replacethe gene encoding ICP4 of a virus also deleted for ICP27 and ICP34.5 andwith an inactivating mutation in the gene encoding VMW65.

[0079] (iv) 1764/27-/4-/pR20.5/vhs

[0080] Virus strain 1764/27-/4-/pR20.5/vhs was constructed by insertionof the pR20.5 cassette into vhs flanking regions at the unique NruI sitein the vhs encoding gene of HSV1 strain 17+ and the resulting plasmid(pR20.5/vhs) was recombined into HSV strain 1764/27-/4- DNA. Virusstrain 1764/27-/4-/pR20.5/vhs is therefore deleted for the genesencoding ICP4, ICP27 and ICP34.5, and has inactivating mutations in thegenes encoding vmw65 and vhs.

[0081] (v) 1764/27-/4-/pR19lacZ

[0082] Virus strain 1764/27-/4-/pR19lacZ was constructed as for virus(iv) above except the pR19lacZ cassette (Wagstaff et al. 1998) wasrecombined into the latency associated transcript (LAT) region of virusstrain 1764/27-/4- rather than the pR20.5 cassette into vhs.

[0083] (vi) 1764/27-/4-/pR20.5/vhs/HBS-Ag

[0084] The lacZ gene in the pR20.5/vhs plasmid was replaced by the geneencoding hepatitis surface antigen (HBS-Ag) by digestion of pHBV130(Gough and Murray, 1982) with XhoI and NsiI and insertion of thefragment released into pSP72 (Promega) between the SalI and SmaI sites.pR20.5/vhs was digested with XbaI and EcoRI to release the lacZ genewhich was replaced by the HBS-Ag gene excised from pSP72 with HindIIIand EcoRI. The resulting plasmid was recombined into1764/27-/4-/pR20.5/vhs viral DNA and non-lacZ expressing plaquesselected and purified. Genome structure was then confirmed by Southernblot.

[0085] Dendritic Cell Preparation

[0086] DC were prepared from peripheral blood as previously described(Coffin et al 1998). Briefly, peripheral blood mononuclear cells (PBMCs)were prepared from 60 ml of healthy/hepatitis B vaccinated donor bloodusing lymphoprep (Nycomed). After removal of red cells, non-adherentcells (mainly T cells and B cells) were removed, washed in HBSS andcentrifuged at 1400 rpm, 5 minutes, RT. The cell pellet was resuspendedin a 2 ml 90% FCS: 10lo dimethylsulphoxide (DMSO) mix, aliquoted andstored at −80 C for subsequent T cell isolation. Adherent cells werecultured in RPMI medium supplemented with GM-CSF (0.1 μg/ml) and IL-4(0.05 μg/ml) and incubated for 7 days, at 37 C, 5% CO₂. After furtherlymphoprep purification cells were then magnetically depleted usinganti-CD19, anti-CD2 (Harlan) and anti-CD3 (Harlan) antibodies and DCwere resuspended in complete RPMI medium for immediate use.

[0087] Isolation of CD4+ T-Cells

[0088] T and B cell frozen as above were defrosted quickly, washed inHBSS and centrifuged at 1400 rpm, for 5 minutes. Cells were resuspendedin 2 ml complete RPMI medium, counted and incubated with anti-CD19(BU12-200 μl neat, Immunology dept, UCL), anti-CD14 (HB246—200 μl neat,Immunology dept, UCL) and anti-HLA-DR (L243—100 μl neat, Immunologydept, UCL) mAb and left on ice for 30 minutes. The cells were washed inHBSS, resuspended in 2 ml complete RPMI medium, mixed with sheepanti-mouse antibodies bound to magnetic beads (Dynabeads, Dynal) at aratio of 10 μl beads/10⁶ contaminating cells and incubated on a rotormixer at 4 C, for 45 minutes. CD4+ T cells were then depleted byremoving the supernatant after placing the cell suspension/magnetic beadmix in contact with a magnet, for 10 minutes, on ice. CD4+ T cells werecounted, resuspended in complete RPNI medium at the appropriateconcentration, left on ice or cultured o/n at 37 C, 5% CO₂ forsubsequent use.

[0089] Infection of DC

[0090] DC were pelleted at 1400 rpm for 5 minutes at room temperature.DC were then infected at MOI of 1 by resuspension in RPMI mediumcontaining virus for 1 hour at 37 C, 5% CO₂. 1 ml of RPMI supplementedwith GM-CSF (0.1 μg/ml) and IL-4 (0.05 μg/ml) was then added and. DCincubated at 37 C, 5% CO₂. For LPS stimulation, RPMI additionallycontaining 10 ng/ml LPS was used.

[0091] Cytokine Analysis

[0092] IL-6, and TNF-α were measured in DC culture supernatants usingcommercially available ELISA kits (R&D Systems). Prior to ELISA,supernatants were collected 42 hours post-infection of DC with theindicated viruses and stored at −20 C before use.

[0093] T Cell Proliferation Assays

[0094] DC and CD4+ T cells were isolated and treated as above fromhepatitis B vaccinated and un-vaccinated human individuals. DC were usedat dilutions from 1×10⁵ DC/ml to 1×10⁴ DC/ml and CD4+ T cells at 1×10⁶cells/ml. Experiments at each of DC concentration were performed intriplicate. 100 μl of DC and 100 μl of CD4+ T cells were added to eachassay well. Where indicated recombinant hepatitis B surface antigen(Austral) was added to wells at a final concentration of 1 μg/well.HSV-1-infected and uninfected DC were cultured with CD4+ T cells for 6days at 37 C, 5% CO₂. 1 μCu/well [³H] thymidine (Amersham) was thenadded and 18 hours later cells harvested and [³H] thymidineincorporation counted.

Example 1 Preliminary Data Showing that HSV Strains not Containing aFunctional vhs Gene give Enhanced Activation of Dendritic CellsFollowing Virus Infection

[0095] Here in each case 1×10⁵ dendritic cells were infected with eachof the viruses by gentle pelleting, resuspension in about 100 μl virussuspension in DMEM, incubation at 37° C. for 1 hr, and transfer into 24well plates with 2 ml of RPMI/10%FCS+100 ng/ml GM-CSF, 50 ng/ml IL-4.These plates were then incubated at 37° C./5% CO₂ overnight. Dendriticcells were also treated with lipopolysaccharide (LPS) a known dendriticcell activator, and untreated as a controls.

[0096] Supernatants from these infections and from the control were thenused in ELISA tests to detect levels of secreted cytokines. Fluorescenceactivated cell sorting (FACS) was also used to detect levels ofexpression of CD86 on the surface of infected and control dendriticcells. In dendritic cell cultures there are two populations of cellswith respect to levels of CD86 expression. These are observed as twopeaks by FACS analysis reflecting a first peak of cells with arelatively lower level of CD86 expression and a second peak of cellswith a relatively higher level of CD86 expression. On activation by e.g.LPS more of the cells express higher levels of CD86 and there are thusmore cells are found in the second peak. TABLE 1 Cytokine concentrationin culture supernatants 24 hr after infection with the indicated virusesor in control supernatants at an MOI of 1. Measured by ELISA. CytokineConcentration (ng/well) Treatment IL-6 TNFa 17+/pR20.5/UL43 6 1.11764/27−/4−/pR191acZ 4 0.8 1764/27−/4−/pR20.5/vhs 46 7.1 No infection 40.9

[0097] Table 1: Cytokine concentration in culture supernatants 24 hrafter infection with the indicated viruses or in control supernatants atan MOI of 1. Measured by ELISA. TABLE 2 Expression of CD86 on controlcells and cells infected with the indicated viruses. % cells % of cellsMean fluorescence Treatment peak 1 peak 2 intensity peak 217+/pR20.5/UL43 43.35 46.85 5 × 10² 17+/pR20.5/UL43 + LPS 56.93 29.061764/27−/4−/pR191acZ 24.1 68.5 5 × 10² 1764/27−/4−/pR191acZ + 52.7135.32 7 × 10² LPS 1764/27−/4−/pR20.5/vhs 27.81 64.61 1 × 10³1764/27−/4−/pR20.5/vhs + 39.52 52.40 9 × 10² LPS No infection 48.9531.45 1 × 10³ No infection + LPS 30.33 60.81 9 × 10²

[0098] Conclusions

[0099] The results show that untreated dendritic cells secrete minimallevels of the cytokines tested and have the expected “resting” levels ofCD86 on their surface. Following LPS treatment cytokine levels aresignificantly stimulated and the level of surface expression of CD86increases significantly. In the experiments above the mean fluorescenceintensity in LPS treated cells is approximately 1×10³ by FACS analysiswith an anti-CD86 antibody. These results are indicative that dendriticcells are in an activated state.

[0100] Following infection of dendritic cells with the indicated virusesit can clearly be seen that for activation of dendritic cells to occurby these assays the virus must contain an inactivating mutation in thegene encoding vhs. Viruses of varying levels of disablement have beenused, and only the virus containing the vhs mutation gives significantactivation of the dendritic cells by these assays. It can also be seenthat if mutation to vhs is not included when cells are treated with LPSas well as infected with the indicated viruses, CD86 levels are notincreased in as many cells as occurs by treatment with LPS alone. Also,unless vhs mutation is included, the mean fluorescence intensity of CD86expressing cells as measured by FACS following virus infection isreduced from that seen if cells are treated with LPS. For maximum immunestimulation by dendritic cells it can thus be concluded thatinactivating mutation(s) in the gene encoding vhs should be included.

Example 2 HSV Strains not Containing a Functional vhs Protein do notBlock Dendritic Cell Activation

[0101] Fluorescence activated cell sorting (FACS) was used to detectlevels of expression of CD86, CD80, CD83 and CD40 on the surface ofinfected and control dendritic cells. Supernatants from the infectionswas used to assess levels of cytokines by ELISA.

[0102] Results

[0103] The ELISA results (FIG. 3) show that while DC can be infectedwith HSV at high efficiency, cytokines indicative of DC activation arenot produced with either a wild type (strain 17+) or a disabled (strain1764/27-/4-) virus. However if vhs is inactivated from strain1764/27-/4- , giving strain 1764/27-/4-/pR20.5/vhs, cytokines indicativeof DC activation are then produced FACS analysis (FIG. 2) on non-LPSstimulated DC shows that infection with essentially wild type HSV(strain 17+) or a replication incompetent HSV vector (strain1764/27-/4-) prevents the increased expression of CD86. As discussedabove, increased CD86 expression would be expected if DC had becomeactivated by the infection process. CD40 levels are also altered/reducedin HSV infected cells. However, if vhs is inactivated (strain1764/27-/4-/pR20.5/vhs), CD86 levels are increased indicatingactivation, and CD40 levels are unaffected. CD80 and CD83 are notgreatly affected in unstimulated DC infected with HSV. CD83 (B7.1) andCD86 (B7.2) are two key T-cell co-stimulatory molecules, CD40 is a keyT-cell activator, and CD83 is a DC marker up-regulated during DCmaturation and activation.

[0104] When DC are LPS stimulated at the time of infection effects onCD40 levels are more marked with both wild type (strain 17+) or disabled(strain 1764/27-/4-) virus. If vhs is inactivated, however, theseeffects on CD40 are prevented. LPS stimulated DC usually significantlyup-regulate CD83 and CD86 expression, but this is blocked by HSV(strains 17+ and 1764/27-/4-) unless vhs is inactivated (strain1764/27-/4-/pR20.5/vhs). When vhs is inactivated, both CD83 and CD86levels are increased to a similar or greater extent as in LPS stimulatedbut uninfected cells.

[0105] Conclusion

[0106] As in the preliminary experiments (Example 1), it can clearly beseen that for dendritic cells to become activated as measured by surfacemarker expression levels in response to HSV infection or HSV infectionand LPS stimulation it is clear that the gene encoding vhs must beinactivated. Viruses encoding functional vhs do not allow dendriticcells to become significantly activated as measured by the expressionlevels of the surface markers tested.

Example 3 DC Transduced with a vhs Inactivated HSV Vector Direct AntigenSpecific T Cell Responses in vitro

[0107] The results above suggested that HSV vectors in which vhs isinactivated might be used as effective vectors for DC as theinactivating effects of HSV in DC have been prevented. Indeed DCinfected with such HSV mutants appear to be specifically activated inresponse to infection as measured by CD86 up-regulation and thesecretion of certain cytokines. To test whether vhs-inactivated HSVmutants might be used to direct antigen specific immune responsesfollowing the delivery of antigen encoding genes to DC, experiments wereperformed using DC and T-cells prepared from hepatitis B vaccinated andun-vaccinated individuals. Here a virus was first constructed in which ahepatitis B surface antigen (HBS-Ag) expression cassette was insertedinto the vhs encoding gene of the IE gene deficient virus. T-cellproliferation assays were then performed in which DC from vaccinated orun-vaccinated individuals were either untreated, ‘loaded’ with antigenby mixing with recombinant HBS-Ag protein, infected with the controlmarker gene containing vector (1764/27-/4-/pR20.5/vhs at MOI=1),infected with the control vector (1764/27-/4-/pR20.5/vhs at MOI=1) andalso mixed with recombinant HBS-Ag, or infected with the vectorexpressing HBS-Ag (1764/27-/4-/pR20.5/vhs/HBS-Ag at MOI=1). DC were thenmixed with T-cells derived from the same vaccinated or unvaccinatedindividuals respectively, and effects on T-cell proliferation observedin standard T-cell proliferation assays.

[0108] These experiments showed (Fig. ) that while HBS-Ag recombinantprotein and the control HSV vector could induce a small T-cellproliferative response in vaccinated individuals, the HSV responseprobably indicating proliferation of T-cells specific to HSV structuralproteins, and the control vector mixed with recombinant HBS-Ag couldillicit a slightly greater response, the HBS-Ag expressing vector gave asignificantly greater response than any of these. Thus followingdelivery of HBS-Ag directly into DC using an HSV vector a significantand specific T-cell proliferative response was induced which did notoccur following mixing with recombinant antigen alone. HSV vectors withvhs inactivated thus allow the delivery of antigen coding genes to DCsuch that DC retain the ability to stimulate antigen specific T-cellproliferative responses.

[0109] References

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I claim:
 1. A method of stimulating an immune response in a human oranimal subject, which method comprises administering to a subject inneed thereof an effective amount of an attenuated herpes virus which:(i) lacks a functional vhs gene, or a functional equivalent thereof;(ii) lacks a functional gene encoding ICP47, or a functional equivalentthereof; and (iii) comprises a functional UL43 gene, or a functionalequivalent thereof such that dendritic cells are infected with saidvirus.
 2. The method of claim 1, wherein said virus is a herpes simplexvirus 1 or
 2. 3. The method of claim 1, wherein said virus lacks atleast one further functional immediate early gene.
 4. The method ofclaim 3, wherein said immediate early gene is selected from genesencoding ICP0, ICP4, ICP22, ICP27 or functional equivalents thereof. 5.The method of claim 3, wherein said virus lacks both a functional geneencoding ICP27 and a functional gene encoding ICP4.
 6. The method ofclaim 1, wherein said virus comprises a heterologous gene.
 7. The methodof claim 1, wherein said heterologous gene is operably linked to acontrol sequence permitting expression of said heterologous gene in adendritic cell.
 8. The method of claim 1, wherein said heterologous geneencodes a polypeptide of therapeutic use.
 9. The method of claim 1,wherein said heterologous gene encodes a polypeptide selected from thegroup consisting of: a polypeptide, the level of expression of which isincreased in or on the surface of tumour cells as compared to non-tumourcells; a polypeptide which is present in or on the surface of tumourcells but absent from non-tumour cells; a polypeptide capable ofmodifying immune responses; and a polypeptide of parasitic, viral orbacterial origin.
 10. The method of claim 1, wherein said viruscomprises more than one heterologous gene.
 11. The method of claim 1,wherein said virus comprises a heterologous gene or genes capable ofmodulating an immune response.
 12. The method of claim 11, wherein saidheterologous gene encodes a chemokine, cytokine, or co-stimulatorymolecule.
 13. The method of claim 1, wherein said subject is a humansubject.
 14. The method of claim 1, wherein the virus is administered byinjection, by infusion, by an intra- or trans-dermal route, or bybiolistic means.
 15. The method of claim 1, wherein the subject is inneed of treatment of or protection against a pathogenic infection. 16.The method of claim 1, wherein the subject is in need of treatment of orprotection against cancer.